The present invention relates to Gram staining methods, and kits of reagents therefore.
Conventional Gram staining technique subjects the sample (frequently a specimen which has been smeared on a glass slide and dried) to four solutions in sequence:
(1) aqueous Gentian Violet (also called Crystal Violet, Hexamethyl Violet C.I. No. 42555), PA1 (2) aqueous iodine-iodide (conventionally KI and I.sub.2 in water), PA1 (3) decolorizer (conventionally acetone admixed with ethanol or isopropanol), and PA1 (4) counterstain (conventionally Safranin solution). PA1 (a) staining the biological specimen with Gentain Violet, and PA1 (b) contacting the Gentain Violet stained specimen with a solution comprising iodine, an iodide salt and an alcohol solvent for a time sufficient to intensify the staining of Gram Positive bacteria and to decolorize Gram Negative bacteria. PA1 (c) counterstaining Gram Negative bacteria in the specimen with a Safranin solution. PA1 (a) an aqueous Gentian Violet solution, and PA1 (b) an alcoholic solution of iodine and an iodide salt. PA1 (c) an aqueous Safranin solution.
Handling of the iodine-iodide solution has been improved by the use of polyvinylpyrrolidone-iodine. Theories about how this technique distinguishes Gram Positive from Gram Negative bacteria have varied, but it is often stated that iodine forms a complex with Gentian Violet that is trapped by a barrier in Gram Positive cells that have been dehydrated and treated with mordant and iodine. In Gram Negative bacteria, the barrier is more penetrable, so that the solvent (decolorizer) extracts the iodine-Gentian Violet complex. Thus, it is a common recommendation to leave the iodine solution on the slide for at least one minute and then remove it by gently rinsing with cold tap water before introducing the decolorizer.